Discovery of three cycloviruses in fecal samples from silver-haired bats (Lasionycteris noctivagans) in Arizona (USA).

Bats harbour a diverse array of viruses, some of which are zoonotic, and are one of the most speciose groups of mammals on earth. As part of an ongoing bat-associated viral diversity research project, we identified three cycloviruses (family Circoviridae) in fecal samples of silver-haired bats (Lasionycteris noctivagans) caught in Cave Creek Canyon of Arizona (USA). Two of the three identified genomes represent two new species in the genus Cyclovirus. Cycloviruses have been found in a wide range of environments and hosts; however, little is known about their biology. These new genomes of cycloviruses are the first from silver-haired bats, adding to the broader knowledge of cyclovirus diversity. With continuing studies, it is likely that additional viruses of the family Circoviridae will be identified in Arizona bat populations.

Bats (order Chiroptera) are one of the most abundant and diverse groups of mammals, with 1,448 extant species currently recognized [1, 2]. Bats are associated with an abundant virome and have primarily been studied from the perspective of zoonotic transmission, with an emphasis on coronaviruses, filoviruses, paramyxoviruses, and rhabdoviruses [3-5]. Over the last decade, there has been a significant interest in analyzing bat-associated viruses using viral metagenomic approaches, which has resulted in the identification of numerous novel and known DNA and RNA viruses [4-6]. However, limited discovery work has been done on bat-associated viruses in Arizona [7-9], even through southern Arizona harbors the greatest diversity of bats of any comparably sized region in the United States of America [2].

Our field research was conducted in Madrean evergreen-woodland on Cave Creek in the Chiricahua Mountains (Cochise County, Arizona). To capture bats, we deployed mist nets (2.6, 4, and 6 m; Avinet Inc., Portland, ME) across areas of calm water on sections of Cave Creek where the tree canopy forced bats into corridors. We recorded species, time of capture, sex, length of right forearm (mm), and weight (g), and collected fecal samples from each bat. All bats were released where they we caught within 15 minutes of capture. On 10 June 2018, we captured 24 bats representing 11 species in mist nets deployed from 19:30 (MST) to midnight. Among those captures were three nonreproductive adult male silver-haired bats (Lasionycteris noctivagans), an insectivorous species characterized by blackish to dark brown pelage with a frosting of whitish silver on the back, two upper premolars, short rounded ears, and a dorsally furred interfemoral membrane [10].

Fecal samples collected from each individual silver-haired bat were pooled and processed as described by Male et al. [12]. In brief, the fecal samples were resuspended in 1 ml of SM buffer and homogenized. The homogenate was then filtered sequentially through 0.45- and 0.2-µm syringe filters. Two hundred µl of the filtrate was then used to extract viral DNA using a High Pure Viral Nucleic Acid Kit (Roche Diagnostics, USA). We amplified circular DNA by rolling-circle amplification (RCA), using a TempliPhi 100 Amplification Kit (GE Healthcare, USA), and the amplified DNA was then used to generate an Illumina sequencing library using a TruSeq Nano DNA kit. The library was sequenced on a NovaSeq6000 sequencer at Psomagen Inc. (USA). The resulting raw reads were trimmed using Trimmomatic v0.39 [13] and then assembled de novo using metaSPAdes v3.14.0 [14]. The de novo-assembled contigs were analyzed using BLASTx (BLAST version 2.11.0) [15] against a viral Refseq protein database (downloaded September 2021). We identified three contigs with circovirus-like sequences (760, 1047, and 2320 nt). In addition, we also identified contigs with similarities to DNA bacteriophages of the family Microviridae (n = 1; 4404 nt) and those formerly assigned to the families Myoviridae (n = 72, 769–7771 nt), Podoviridae (n = 3; 1119–7008 nt), and Siphoviridae (n = 51; 767–3265 nt).

In this report, we focus on the three circovirus-like contigs for which, based on the de novo-assembled contigs, we designed abutting primer pairs (Table 1) in order to recover these full genomes. The RCA product was used as a template with the specific primer pairs (Table 1) to amplify the full genomes of the three circovirus-like sequences using Kapa HiFi DNA polymerase (Roche Diagnostics, USA) according to the manufacturer’s recommendations. The amplicons were resolved by electrophoresis in a 0.7% agarose gel, purified, and cloned into the vector pJET 1.2 (Thermo Fisher Scientific, USA). Competent XL1-Blue Escherichia coli cells were transformed with the recombinant plasmids, which were then sequenced by the Sanger method at Macrogen Inc. (South Korea), using primer walking. The Sanger sequences were assembled using Geneious Prime 2021.0.3 (Biomatters Ltd., New Zealand). Open reading frames (ORFs) were identified using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/).

Table 1

Summary of the primers used to recover the complete genomes of the three cycloviruses, with their GenBank accession numbers and genome lengths as well as their nonanucleotide, HUH endonuclease, and superfamily 3 helicase motifs

					HUH endonuclease			Superfamily 3 helicase
Virus	Primer pair	Accession no.	Genome length (nt)	Nonanucleotide	Motif I	Motif II	Motif III	Walker A	Walker B	Motif C
Chifec virus UA15_35	F: AGATCGTGTTCATCGCCATCGTTATAGTT R: TAAAGCAAGACTGGGTGACACTGTCGTTT	OM262453	1758	AAGTATTAC	CFTKNN	RHLQGY	QNLTYCSK	GPPGTGKSRRFA	IIDDF	FISSN
Chifec virus UA15_517	F: TTGTACAATTCGTCGGTATTGATGTTAGAG R: ATTTGAAGGTTATGTAAACACAGCATTCGA	OM262454	1779	TAGTATTAC	VYTLNN	PHLQGF	DNQKYCSK	GEPGTGKSRTAL	IIDDF	WITSN
Chifec virus UA15_2320	F: GAGAGGATTTTGAGCAATAGTCCTTGTTTT R: AGTTTGAAGAATTTGGAATCCTACCAAGTC	OM262459	2320	TAGTATTAC	CWTKNN	RHLQCY	QNKDYCSK	GPTRTGKTRLAA	VLDDY	IITSN

A BLASTn web search against the NCBI nt database of the three complete Sanger-sequenced genomes (1758–2320 nt; OM262453, OM262454, OM262459) revealed that they shared the highest similarity with various cycloviruses of the family Circoviridae. Circoviridae is a family of single-stranded circular DNA viruses with an ambisense genome organization and two ORFs coding for the capsid protein (Cp) and replication-associated protein (Rep) [16]. There are two genera within this family, Circovirus and Cyclovirus [16]. Unlike members of the genus Circovirus that have been implicated in various diseases in mammals and birds (e.g., postweaning multisystemic wasting syndrome in pigs and psittacine beak and feather disease in parrots), relatively little is known about members of the genus Cyclovirus [17]. A feature that distinguishes the genomes of circoviruses and cycloviruses is the orientation of the rep and cp genes relative to the conserved nonanucleotide motif. The rep gene is on the virion sense strand, whereas the cp gene is on the complementary strand for members of the genus Circovirus and vice versa for those of the genus Cyclovirus [17]. Although cyclovirus genomes have been identified in various environmental and animal samples [17], including those obtained from members of the orders Anseriformes (n = 1), Araneae (n = 2), Artiodactyla (n = 7), Blattodea (n = 1), Carnivora (n = 52), Chiroptera (n = 29), Diptera (n = 1), Eulipotyphla (n = 7), Galliformes (n = 11), Hymenoptera (n = 3), Odonata (n = 35), Passeriformes (n = 10), Perissodactyla (n = 1), Primates (n = 27), Rodentia (n = 7), and from a plant of the order Fabales (n = 1), no definite hosts have been identified so far, and thus, their biology is unknown.

In the three cyclovirus genomes from silver-haired bats, we identified a conserved nonanucleotide motif, “NAGTATTAC”. Additionally, in the Rep sequence, we identified RCR endonuclease and superfamily 3 (SF3) helicase motifs (Table 1). To determine the phylogenetic relationship of the viruses to other cycloviruses, 195 genome sequences of cycloviruses were downloaded from the GenBank database on 10 Jan 2022 and aligned with those from silvered-haired bats identified in this study. Reverse complement genome sequences of two circoviruses (porcine circovirus 1 and 2) were used as an outgroup. These were aligned using MAFFT v7.113 in AUTO mode [18], and the resulting alignment was used to infer a maximum-likelihood phylogenetic tree using PhyML 3.0 [19] with the GTR + I + G substitution model. Branches with less than 60% bootstrap support were collapsed using TreeGraph2 [20]. The phylogenetic tree was visualized and edited in iTOL v6 [21]. The three cyclovirus genomes from silvered-haired bats share ~ 57–60% genome-wide pairwise identity (calculated using SDT v1.2 [22]) and are phylogenetically distinct from each other (Fig. 1; Supplementary Data 1). Chifec virus UA15_517 (OM262454) shares 87% genome-wide pairwise identity with dragonfly cyclovirus 6 (KC512918), which is the sole known member of the species Dragonfly associated cyclovirus 6 (Supplementary Data 1). Members of the family Circoviridae are classified into species based on their genome-wide pairwise identity with a species demarcation threshold of 80% [17]; thus, chifec virus UA15_517 would be a member of the species Dragonfly associated cyclovirus 6. Chifec virus UA15_35 (OM262453) shares < 61% identity with all other cycloviruses and clusters phylogenetically with members of the species Horse associated cyclovirus 1 (Fig. 1; Supplementary Data 1). Chifec virus UA15_2320 (OM262459) shares < 70% identity with all other cycloviruses and clusters phylogenetically with Caesalpinia ferrea associated virus (MT707947) and members of the species Bat associated cyclovirus 7 and Mouse associated cyclovirus 1 (Fig. 1; Supplementary Data 1). Chifec virus UA15_35 and UA15_2320, based on the species demarcation threshold, each represent a new species of cycloviruses.

Fig. 1

Maximum-likelihood phylogenetic tree inferred from the alignment of the genome sequences of cycloviruses and rooted with the reverse completement sequences of members of the genus Circovirus. The source at the level of order (Anseriformes, Araneae, Artiodactyla, Blattodea, Carnivora, Chiroptera, Diptera, Eulipotyphla, Galliformes, Hymenoptera, Odonata, Passeriformes, Perissodactyla, Primates, Rodentia and Fabales) of the genomes is shown in color-coded boxes. Branches with more members within a species have been collapsed. Accession numbers are provided for unclassified cycloviruses and for species that only has a single member. The phylogenetic tree was rooted with reverse complement genome sequences of representative members of the genus Circovirus

The three cycloviruses identified in the fecal samples of silver-haired bats are the first from this bat species, and only two other cycloviruses from bats have been described in the USA, one from the pallid bat (Antrozous pallidus) [23] and one from the Mexican free-tailed bat (Tadarida brasiliensis) [24]. Besides these, other cycloviruses have been identified in bat guano and tissue from bats of various species (Chalinolobus gouldii, Molossus molossus, Myotis spp., Nyctalus noctula, Pipistrellus nathusii, Plecotus auritus, Pteropus tonganus, Rhinolophus ferrumequinum, Rhinolophus pusillus, Tadarida brasiliensis, Tylonycteris pachypus, Vespertilio superans) from Australia, Brazil, China, Hungary, Tonga, and Ukraine [12, 25–28]. Since we detected the cycloviruses in bat feces, we are unable determine whether these are diet-related viruses or ones that truly infect silver-haired bats. Examination of feces across their range has shown that silver-haired bats feed on a variety of insects, including representatives of the orders Lepidoptera, Hemiptera, Coleoptera, Diptera, and Trichoptera [10, 11]. Thus, it is likely that the cycloviruses identified in silver-haired bat feces infect these insects. Nonetheless, given the increased focus on bats as reservoirs of various viruses, it is likely that the identification of novel cycloviruses in bat samples will continue.

Below is the link to the electronic supplementary material

Supplementary Material 1